title: Diazepam enhancement of GABA-gated currents in binary and ternary GABAA receptors: relationship to benzodiazepine binding site density.
abstract: Although the predominant GABAA receptor isoform in the adult rodent central nervous system is a ternary complex composed of alpha 1 beta 2/3 gamma 2-subunits, small populations of binary receptors lacking beta-subunits (i.e., complexes containing alpha gamma-subunits) have also been identified. When expressed in HEK 293 cells, recombinant GABAA receptors composed of either alpha 1 beta 2/3 gamma 2- or alpha 1 gamma 2-subunits form benzodiazepine-responsive, GABA-gated chloride channels. The objective of this study was to compare the ability of a prototypic benzodiazepine (diazepam) to augment GABA-gated chloride currents in these binary and ternary receptor isoforms. The potency of GABA was characteristically increased by diazepam (1 microM) in both receptor isoforms, but this increase was significantly greater (p < 0.05) in receptors composed of alpha 1 beta 2 gamma 2-subunits (approximately five- to sixfold) compared to alpha 1 gamma 2-subunits (approximately 2.2-fold). At GABA concentrations approximating its EC50 value (5 microM), the greater augmentation observed in ternary receptors was attributable to a higher efficacy of diazepam. Radioligand binding studies revealed that the Bmax of [3H]flunitrazepam was increased approximately 1.8- and 3.5-fold in cells expressing alpha 1 beta 2 gamma 2- and alpha 1 beta 3 gamma 2-subunits, respectively, compared to cells expressing alpha 1 gamma 2-subunits. A similar increase (approximately 3.8-fold) in the Bmax of [3H]Ro 15-4513 was observed in HEK 293 cells transiently transfected with cDNAs encoding alpha 6 beta 3 gamma 2-compared to alpha 6 gamma 2-subunits. The Kd values of these radioligands were not different in binary and ternary receptor isoforms. It is hypothesized that the greater efficacy of diazepam in alpha 1 beta 2 gamma 2 compared to alpha 1 gamma 2 GABAA receptors results from the higher benzodiazepine binding site density produced by the formation of a ternary complex.
authors.affiliation: Laboratory of Neuroscience, National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, MD 20892, USA.
authors.forename: R
authors.initials: R
authors.lastname: Granja
authors.forename: D
authors.initials: D
authors.lastname: Gunnersen
authors.forename: G
authors.initials: G
authors.lastname: Wong
authors.forename: A
authors.initials: A
authors.lastname: Valeyev
authors.forename: P
authors.initials: P
authors.lastname: Skolnick
mesh_terms: D000818:Animals; D001569:Benzodiazepines / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D002460:Cell Line; D018118:Chloride Channels / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D003975:Diazepam / Q000378:metabolism / Q000494:pharmacology*; D005445:Flunitrazepam / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects* / Q000502:physiology; D007668:Kidney / Q000166:cytology; D008564:Membrane Potentials / Q000187:drug effects; D011869:Radioligand Assay; D051381:Rats; D011963:Receptors, GABA-A / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D005680:gamma-Aminobutyric Acid / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*
chemical_list: D018118:Chloride Channels; D011963:Receptors, GABA-A; D001569:Benzodiazepines; D005680:gamma-Aminobutyric Acid; D005445:Flunitrazepam; D003975:Diazepam